It is imperative that serious Staphylococcus aureus diseases such as bacteremia and infectious endocarditis are treated aggressively with effective antimicrobial agents. These diseases had mortality rates of 80 and 100 percent respectively in the pre-antibiotic era. Most hospital strains of S. aureus are now methicillin-resistant (MRSA), and such strains are typically resistant to multiple other antibiotics. The glycopeptide antibiotic vancomycin was the sole remaining antibiotic to which S. aureus remained uniformly susceptible. Recently, vancomycin-resistant strains have arisen in patients during long-term vancomycin therapy (so called glycopeptide-intermediate susceptible S. aureus or GISA strains). It is imperative that we understand the mechanism of vancomycin resistance in such strains. Vancomycin-resistant strains have been step-selected in the investigators laboratory. It is likely that the resistance mechanism involves several mutations and is multifaceted. The investigators propose to study the mechanisms of S. aureus vancomycin and methicillin resistance in laboratory and clinical strains, and their work is organized into three major lines of investigation: I. Phenotypic studies of vancomycin resistance; II. Genotypic studies of vancomycin resistance; and III. Stress gene and protein expression in methicillin and vancomycin resistance. In I, they will study the composition and structure of peptidoglycan, teichoic acid and lipoteichoic acid in GISA strains, and attempt to understand the mechanism of decreased autolytic activity observed in such strains. They will attempt to understand the increased NaCl-sensitivity of GISA strains through studying the accumulation of compatible solutes, and whether Na+ ions accumulate intracellularly to growth inhibitory levels upon NaCl stress. In II, they will attempt to discover the genetic basis of vancomycin resistance through transposon mutagenesis studies, direct cloning of genes responsible for vancomycin resistance, and cloning and nucleotide sequence analysis of target genes as indicated to play a role in vancomycin resistance through studies in I above. In III, they will study the role of cell wall antibiotic stress gene and protein expression in methicillin and vancomycin resistance through a proteomics approach, transcription profiling using gene chip technology, and by selective capture of transcribed sequences. It is expected that these studies will lead to improved methods for the control of methicillin-resistant and vancomycin-resistance S. aureus infections, and form the basis for development of novel antistaphylococcal agents.